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Water column mixing homogenizes thermal and chemical gradients which are known to define distribution of microbial communities and influence the prevailing biogeochemical processes. Little is however known about the effects of rapid water column mixing on the vertical distribution of microbial communities in stratified reservoirs. To address this knowledge gap, physicochemical properties and microbial community composition from 16 S rRNA amplicon sequencing were analyzed before and after mixing of vertically stratified water-column bioreactors. Our results showed that α-diversity of bacterial communities decreased from bottom to surface during periods of thermal stratification. After an experimental mixing event, bacterial community diversity experienced a significant decrease throughout the water column and network connectivity was disrupted, followed by slow recovery. Significant differences in composition were seen for both total (DNA) and active (RNA) bacterial communities when comparing surface and bottom layer during periods of stratification, and when comparing samples collected before mixing and after re-stratification. The dominant predicted community assembly processes for stratified conditions were deterministic while such processes were less important during recovery from episodic mixing. Water quality characteristics of stratified water were significantly correlated with bacterial community diversity and structure. Furthermore, structural equation modeling analyses showed that changes in sulfur may have the greatest direct effect on bacterial community composition. Our results imply that rapid vertical mixing caused by episodic weather extremes and hydrological operations may have a long-term effect on microbial communities and biogeochemical processes.

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The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

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Drug delivery systems (DDSs) have great potential for improving the treatment of several diseases, especially microbial infections and cancers. However, the formulation procedures of DDSs remain challenging, especially at the nanoscale. Reducing batch-to-batch variation and enhancing production rate are some of the essential requirements for accelerating the translation of DDSs from a small scale to an industrial level. Microfluidic technologies have emerged as an alternative to the conventional bench methods to address these issues. By providing precise control over the fluid flows and rapid mixing, microfluidic systems can be used to fabricate and engineer different types of DDSs with specific properties for efficient delivery of a wide range of drugs and genetic materials. This review discusses the principles of controlled rapid mixing that have been employed in different microfluidic strategies for producing DDSs. Moreover, the impact of the microfluidic device design and parameters on the type and properties of DDS formulations was assessed, and recent applications in drug and gene delivery were also considered.

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In-plant wastewater treatment strategies to deal with bypass wastewater in excess of plant capacity are critical in securing sustainable wastewater management. To address this issue, potassium ferrate(VI), which is a dual disinfectant and coagulant, is assessed in this study as the sole chemical applied to enhance the primary treatment of bypass wastewater. The effect of rapid mixing speed is investigated for the first time along with potassium ferrate(VI) dosage by means of central composite design and response surface methodology. Escherichia coli (E. Coli), Faecal Coliform (FC), Total Suspended Solids (TSS), and Orthophosphates ( P O 4 3 - ) were considered as the process responses. All responses other than P O 4 3 - showed good agreement between the observed and modelled values. While there was no point of maximum or minimum response for both E. Coli and FC, whose removals were found to increase with the increase of both the mixing intensity and potassium ferrate(VI) dosages, TSS removal exhibited optimal responses. The effluent quality achieved by potassium ferrate(VI), as an independent treatment, can be sufficient for certain types of unrestricted and restricted irrigation reuse purposes suggested by World Health Organisation (WHO) reuse guidelines.

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Stopped-flow spectroscopy is a powerful method for measuring very fast biological and chemical reactions. The technique however is often limited by the volumes of reactants needed to load the system. Here we present a simple adaptation of commercial stopped-flow system that reduces the volume needed by a factor of 4 to ≈120 µl. After evaluation the volume requirements of the system we show that many standard myosin based assays can be performed using <100 µg of myosin. This adaptation both reduces the volume and therefore mass of protein required and also produces data of similar quality to that produced using the standard set up. The 100 µg of myosin required for these assays is less than that which can be isolated from 100 mg of muscle tissue. With this reduced quantity of myosin, assays using biopsy samples become possible. This will allow assays to be used to assist diagnoses, to examine the effects of post translational modifications on muscle proteins and to test potential therapeutic drugs using patient derived samples.

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HYPOTHESIS: In conventional 'bulk' nanoprecipitation, the capacity to load hydrophobic drugs into the polymeric nanoparticles (NPs) is limited to about 1%. The size distribution of the resulting NPs becomes polydisperse when higher precursor concentration is used to increase the drug loading. Hence, it should be possible to enhance the hydrophobic drug loading in polymeric NPs while maintaining the uniform NP size distribution by optimizing the nanoprecipitation process and purification process. EXPERIMENTS: Systematic studies were performed to enhance the loading of docetaxel (Dtxl) by using a process of centrifugal spin-down, rapid mixing by turbulence, and addition of co-solvent. The size distributions and Dtxl loading of the NPs were measured using dynamic light scattering and HPLC, respectively. FINDINGS: The centrifugal spin-down process helps to maintain uniform size distribution even at the high precursor concentration. In bulk nanoprecipitation, the resulting NPs achieved Dtxl loading up to 3.2%. By adopting turbulence for rapid mixing, the loading of Dtxl increased to 4.4%. By adding hexane as co-solvent, the loading of Dtxl further increased to 5.5%. Because of the drug loading augmentation, high degree of control, and extremely high production rate, the developed method may be useful for industrial-scale production of personalized nanomedicines by nanoprecipitation.

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In this chapter, we introduce a nanodisc-based experimental platform to study Ca2+-triggered membrane interaction of synaptotagmin-1. We describe and discuss in detail how to assemble this soluble mimetic of the docked vesicle-plasma membrane junction, with fluorescently labeled synaptotagmin-1 bound to trans SNAREpins assembled between nanodiscs and present the stopped-flow rapid mixing method used to monitor the conformational dynamics of Ca2+-activation process on a millisecond timescale.

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The free-standing polyaniline (PANI)-based composite film electrodes were prepared with polyvinyl chloride (PVC) and the aniline modified PVC (PVC-An) films as flexible substrates for supercapacitors, via facile in-situ chemical oxidative polymerization of aniline, with conventional chemical oxidative polymerization or rapid-mixing chemical oxidative polymerization technique. Owing to the grafting of PANI from the PVC-An film as substrate and the suppression of the secondary growth of the primary PANI particles in the rapid-mixing chemical oxidative polymerization, the PVC-g-PANI-2 composite film with loose surface possessed better comprehensive performance, accompanying the high specific capacitance (645.3F/g at a current density of 1A/g), good rate capacitance (retaining 63.2% of original value at a current density of 10A/g and 52.0% at a scan rate of 100mV/s), good cycle stability (retaining 83.1% after 1000 cycles) and the improved internal resistance. Besides its excellent flexibility, it could retain 61.2% of its original specific capacitance under the stress of 8.66MPa for 1h, demonstrating a good tensile-resistance.

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In the folding of ß-lactoglobulin (ßLG), a predominantly ß-sheet protein, a transient intermediate possessing an excess amount of non-native α-helix is formed within a few milliseconds. To characterize the early folding dynamics of ßLG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native α-helix formation within 200µs. Time-resolved SAXS showed that the radius of gyration (R(g)) of the intermediate at 300 µs was 23.3±0.7 Å, indicating a considerable collapse from the unfolded state having R(g) of 35.1±7.1 Å. Further compaction to R(g) of 21.2±0.3 Å occurred with a time constant of 28±11 ms. Pair distribution functions showed that the intermediate at 300 µs comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of α-helix but similar R(g) of 23.0±0.8 Å, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native α-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of ßLG.

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Time series data can provide valuable insight into the complexity of biological reactions. Such information can be obtained by mass-spectrometry-based approaches that measure pre-steady-state kinetics. These methods are based on a mixing device that rapidly mixes the reactants prior to the on-line mass measurement of the transient intermediate steps. Here, we describe an improved continuous-flow mixing apparatus for real-time electrospray mass spectrometry measurements. Our setup was designed to minimize metal-solution interfaces and provide a sheath flow of nitrogen gas for generating stable and continuous spray that consequently enhances the signal-to-noise ratio. Moreover, the device was planned to enable easy mounting onto a mass spectrometer replacing the commercial electrospray ionization source. We demonstrate the performance of our apparatus by monitoring the unfolding reaction of cytochrome C, yielding improved signal-to-noise ratio and reduced experimental repeat errors.

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Electron transfer (ET) reactions within proteins are accomplished by a broad set of redox-active molecules, including natural amino acids. Tryptophan participates in ET chemistry as both a cation and a neutral radical. Identification and characterization of the biologically relevant species is essential to understand efficient ET mechanisms in proteins. We present resonance Raman spectra and excitation profiles of the tryptophan cation radical generated by combining a strong oxidant, Ce(IV), with tryptophan model compounds in a fast-flow mixing device. Isotopically modified derivatives, coupled with calculations, allowed the assignment of the normal modes of this radical. Raman bands that are sensitive to protonation state and hydrogen bonding environment of the cation radical were identified. The present findings, along with resonance Raman spectra of the closed-shell and neutral radical counterparts, form a foundation for probing tryptophan-mediated ET reactions in proteins.